Engineering Yarrowia lipolytica to Produce l‑Malic Acid from Glycerol

The\ndeclining availability of cheap fossil-based resources has\nsparked growing interest in the sustainable biosynthesis of organic\nacids. l-Malic acid, a crucial four-carbon dicarboxylic acid,\nfinds extensive applications in the food, chemical, and pharmaceutical\nindustries. Synthetic biology and metabolic engineering have enabled\nthe efficient microbial production of l-malic acid, albeit\nnot in Yarrowia lipolytica, an important\nindustrial microorganism. The present study aimed to explore the potential\nof this fungal species for the production of l-malic acid.\nFirst, endogenous biosynthetic genes and heterologous transporter\ngenes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis\npathway grown on glycerol. Second, overexpression of isocitrate lyase,\nmalate synthase, and malate dehydrogenase in the glyoxylate cycle\npathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent\nincrease to 37.0 g/L was attained through shake flask medium optimization.\nThird, adaptive laboratory evolution allowed the engineered strain <i>Po1g-CEE2+Sp</i> to tolerate a lower pH and to accumulate a\nhigher amount of l-malic acid (56.0 g/L). Finally, when scaling\nup to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion,\nthis study demonstrates for the first time the successful production\nof l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving\nthe way for large-scale sustainable biosynthesis of this and other\norganic acids.